1. Field of the Invention
The present invention relates to a non-immunoaffinity binding method for the detection and assay of aminoglycoside antibiotics in foods of animal origin using lysozyme and .alpha.-lactalbumin as the binding proteins. It also relates to the purification of aminoglycosides from biological fluids such as fermentation broths and to the purification of biological fluids such as milk.
2. Description of the Prior Art
The aminoglycoside antibiotics consist of a group of related antibiotics containing aminosugar residues. The primary effect of the aminoglycoside antibiotics is the inhibition of protein synthesis (Wilhelm et al, Biochem. Vol. 17, 1149-1153, 1978; Wilhelm et al, Biochem., Vol. 17, 1143-1149, 1978). Some of these antibiotics have been approved as a feed additive to control parasitic, respiratory and enteric infections in farm animals. Therefore, rapid methods are needed to screen for the presence of trace levels of these class of antibiotics in biological fluids and tissues. Hygromycin B inhibits the growth of microorganisms and mammalian cells and is used to control parasitic, respiratory and enteric infections in poultry and swine. Residues from hygromycin B may occur in tissues and fluids of farm animals designated for human consumption. Spectinomycin hydrochloride is allowed for treatment and control of bacterial enteritis in swine and in the prevention and control of losses due to chronic respiratory disease associated with M. gallisepticum (PPLO) in poultry. The tolerance for spectinomycin residue is 0.1 ppm in the uncooked edible tissues of chickens. Neomycin residues in eggs are quite stable to normal egg preparation procedures. Frying eggs causes little or no loss of activity, poaching results in a 25% loss, and soft boiling and hard boiling causes little or no loss of activity. Antibiotics administered to dairy cattle whether applied by infusion, injection or oral means, sometimes enters the milk supply. This presence of antibiotics in milk, at the consumer level, can cause allergic reactions, affect starter cultures, or create environments favorable for resistant bacteria (Bishop et al, IN: Standard Methods for the Examination of Dairy Products, 347-395, 1992, 16th Edition, R. T. Marshall, ED., American Public Health Association, Washington, D.C.).
Rapid methods are needed to screen for the presence of trace levels of specific antibiotics or a class of antibiotics in biological fluids and tissues. Enzyme immunoassay techniques have been used only to a limited extent for detection of veterinary drugs due to the lack of sensitivity needed for detection at tolerance or safe levels. The high specificity of immunoassays also limits the number of compounds that can be analyzed compared to a broad spectrum detection obtainable using microbial inhibition assays. Production of antibodies with desired specificities and affinities is time consuming. There are various methods for detection of aminoglycoside antibiotics in biological fluids. Yao et al (J. Antibiotics (TOKYO), Vol.37 (11), 1462-1468, 1984) report a technique useful in the identification, quantification, and screening of aminoglycosides using a heterogeneous microplate enzyme immunoassay with a sensitivity of 10 pg/ml (1 pg/assay) toward gentamicin. Aminoglycoside antibiotics, such as sisomicin, forimicin B, seldomycin, tobramycin, kanamycin A, kanamycin B, spectinomycin, apramycin, hygromycin B, butirosin, streptomycin, paromomycin, validamycin A, sorbistin B, streptothricin, and neomycin A; in fermentation broths were detected by the method. Purified gentamicin antibody is coated onto the surface of wells of a microtiter plate and incubated with gentamicin-alkaline phosphatase conjugate. The amount of enzyme bound to the antibody was quantified by measuring the change in absorbance at 410 nm after the addition of the substrate, p-nitrophenyphosphate. Competitive assay performed by incubating the antibody and enzyme conjugate with various aminoglycosides showed that the antibody probe cross-reacted with all aminoglycosides tested, except neomycins B and C. No cross-reaction was detected with non-aminoglycoside antibiotics.
The analysis of spectinomycin in foods includes chromatographic methods utilizing ion pair solid phase extraction, HPLC separation followed by post column derivatization and fluorescent detection of its 2-naphthalenesulfonyl chloride (NSCl) derivatives. The sensitivity of the liquid chromatographic method is 4 ng per sample load (Tsuji et al, J. Chromatography, Vol. 333, 365-380, 1985). A high performance liquid chromatographic (HPLC) method is reported for detection in turkey plasma at 1.42 ppm (Burton et al, J. of Chromatography, Vol. 571, 209-216,1991) and in swine, chicken, and calf plasma at 50 ppm (Haagsma et al, J. Chromatography, Biomedical Applications, Vol. 615, 289-295, 1993). The tolerance level for spectinomycin residue is 100 ppb in uncooked edible tissues in chickens. The use of Bacillus stearothermophilus and a 65.degree. C. incubation yielded a rapid assay with a sensitivity of 0.2 micrograms per gram of egg (Katz et al, J. Assoc. Off. Anal. Chem., Vol. 61(5), 1103-1106, 1978). The official method for detection of spectinomycin is a microbial turbidimetric assay with an LDL (lowest detectable level) of 2.8 ppm in all tissues of all species (NADA 47-244, New Animal and Drug Application, Upjohn Company, Kalamazoo, Michigan). The analytical range for this assay is 24 to 37.5 ppm. Therefore, a rapid method with higher sensitivity than the microbiological turbidimetric assay is needed to rapidly screen for the presence of spectinomycin at ppb levels.
There are various methods for purification of aminoglycosides from biological fluids. U.S. Pat. No. 4,729,951 (Fereczy et al) discloses a detection and quantification method for determining the presence of aminoglycosides in fermentation broth using thin-layer chromatography. The antibiotic composition is determined by using silica gel as adsorbent and a 1:1:1 mixture of ethanol, methyl-ethyl ketone, and 25 percent aqueous ammonium hydroxide as developing solvent. 0.1 to 0.5 .mu.g/ml portions of the aminoglycoside solution is spotted onto the plates. After drying, a medium containing Bacillus subtilis is layered over the surface of the plate which is subsequently incubated at 37.degree. C. for 16 hours. The size of the inhibition zones is measured and compared to a standard.
U.S. Pat. No. 4,482,707 (Sakakibara et al) discloses a purification method for saccharocin, an aminoglycoside antibiotic, produced by a fermentation process. Culture broth is adjusted to acidic pH, neutralized, and filtered to obtain a filtrate. The active principles are adsorbed on an ion-exchange resin after passing the culture filtrate through a column of the same. The active principles are eluted by 1N aqueous ammonia, and the eluate is concentrated. After adjusting the pH, the concentrate is passed through a column of CM-sephadex C-25 (NH.sub.4.sup.+ type) to adsorb the active principles, and the column is eluted with 0.03N aqueous ammonia.
While there are various methods for detecting aminoglycoside antibiotics in the animals, there still remains a need in the art for a rapid, cost effective detection method. Furthermore, there still remains a need in the art for a rapid, cost effective method for purifying aminoglycosides. Finally, there remains a need in the art for a rapid method for the removal of aminoglycosides from biological fluids such as milk. The present invention is different from prior art methods and provides for a non-immunoaffinity chromatography method for detecting, purifying, and removing aminoglycosides from biological materials.